We have discovered human platelet microbicidal proteins (PMPs)that are microbicidal chemokines, termed human platelet kinoddins (hPKs) to reflect their microbicidal and chemokine functions. hPKs have previously unrecognized microbicidal activity in human blood, but differ markedly in structure from cytotoxic defensin-like peptides. Thus, hPKs have unique structure-activity correlates to confer microbicidal activity withoutconcomitant cytotoxicity. Specific hPK domains confer direct antimicrobial activity, and have amplified microbicidal activity in environments simulating acidic phagolysosomes. Distinct hPK domains promote neutrophil phagocytosis and intracellular killingof pathogens. We hypothesizethat specific determinantsin hPKs govern these independentbut complementary antimicrobial functions. Yet, structural determinants responsible for the coordinatedhost defense roles ofhPKs are unknown. We propose these determinants can be identified, their antimicrobial features defined, and structure-activity signatures resolved. Our complementary Specific Aimswill explorethese hypotheses: Specific Aim 1;Identify the structural determinants governing direct microbicidal functions of hPKs. We will assess peptide libraries of key hPKs to identify domains that confer relevant microbicidal functions. Strategic mutant peptides and synthetic analogues will be used to isolate precise structural determinants responsible for the shared or unique microbicidal profiles of distinct hPKs. Specific Aim 2: Define the roles of specific hPKdomains on keyneutrophil antimicrobial functions. We will dissect hPK domains that potentiate neutrophil phagocytosis, oxidatiye burst, or intracellular killing in vitro using multicolor flow cytometry. These studies will define which hPK domains enhance the key antimicrobial mechanisms in neutrophils directly, or by augmenting the inherent capabilities of these cells. Specific Aim 3;Resolve structure-activity relationships (SARs) in hPKantimicrobial determinants.We will resolve SAR themes in hPK determinants using complementary NMR, CD, and FTIR studies to guide molecular modeling. Unique to our studies will be mosaic peptides designed to validate and interchange the roles of functional signatures in distinct hPKs. These approaches will identify the specific SARs and functional determinants that may be specific to or shared among distinct hPKs or other kinocidins. Our discoveries create a unique opportunity to define molecular signatures in hPKs that govern their multiple antimicrobial functions in innate and adaptive immunity. In turn, these insights will accelerate development of novel strategies to prevent or treat serious infections, particularly those caused by antibiotic-resistant pathogens.